Novel aldosterone intermediates



United States Patent Office 3,211,724 Patented Oct. 12, 1965 3,211,724 NOVEL ALDOSTERONE INTERMEDIATES Tadeus Reichsteiu, Weisseusteiustrasse 22, Basel, Switzerland, and Albert Wettstein, Georg Anner, Jean-Reu Billeter and Karl Heusler, Basel, Robert Neher, Binningeu, Julius Schmidliu, Basel, Hellmut Ueberwasser, Rieheu, and Peter Wieland, Basel, Switzerland; said Wettstein, Auner, Billeter, Heusler, Neher, Schmidlin, Ueberwasser, and Wieland, assignors to said Reichsteiu No Drawing. Filed July 14, 1959, Ser. No. 826,913 Claims priority, application Switzerland, Jan. 15, 1954, 1,038; Feb. 5, 1954, 1,803; Feb. 25, 1954, 2,612; Mar. 5, 1954, 2,942; May 18, 1954, 5,862

16 Claims. (Cl. 260-239.55)

This is a continuation-in-part to Serial No. 480,061, filed January 5, 1955 by Tadeus Reichstein et al., now US. Patent No. 2,904,545.

The highly active hormone of the suprarenal gland which is responsible for the regulation of the mineral metabolism in the human body is known to be a pregnene Aldosterone lNaJOA 01'03 H l Wolfi-Kislmer CH; O O OH 2. CrOa CH COOCH and l 1. CHzNg 3,1l-diketo-al1oetiaulc acid methyl ester compound of the following formulae which obviously represent an equilibrium:

These formulae have been established by a series of degradation reactions which are summarized in the following diagram:

Aldosteroue monoacetate (more readily esterifiable hydroxyl group is acylated) CHgOAC OCO CO Instead of aldosterone, there may be used as starting material functional derivatives thereof in which both hydroxyl groups are free or in which one hydroxyl group is functionally converted, preferably esters, or ethers, enol esters, enol ethers, acetals, or their corresponding thio derivatives, such as thio ethers and thio acetals.

The present invention relates to various intermediate products for the synthesis of aldosterone which products can be obtained by total synthesis or by degradation as indicated above, namely to compounds of the formula in which R is a free or esterified hydroxyl group, a hydroxyl group acetalized with an 18-oxo group or lactonized with an l3-carboxy group or an oxo group, R is a lactonized carboxyl group or an acetalized aldehyde group or a free or esterified hydroxy-methyl group, R is a free or esterified carboxyl group, and X is a free or esterified hydroxyl group, a free or ketalized oxo group, and in which formula there may also be a double bond extending from the 5 carbon atom.

Among these compounds there are for example (18 1l/i)-lactones of A -3-oxo-11p-hydroxy-18-oic acidetienic acids and their esters, (11B 18)-cyclosemiacetals of A -3,18-dioxo-1lp-hydroxy etienic acids and their 20,18-lactones and corresponding functional derivatives, such as 3-ketals and 3 esters as well as the corresponding compounds saturated in the nucleus. There may also be mentioned the 18,11-lactone of 3 13,11,3-dihydroxy-alloetienic acid-18-acid and its 3/3-acetate.

In copending application Serial No. 769,996, now US Patent No. 3,014,903, filed October 28, 1958 by Tadeus Reichstein et al., which is a continuation in part to application Serial No. 585,144, filed May 16, 1956 and now abandoned, which is in turn a continuation-in-part to Serial No. 480,062, filed January 5, 1955, now abandoned, there is described a process for synthesizing compounds of the general formula the I -oo-n in which R is a hydrogen atom, a methyl group or a free or esterified hydroxy methyl group and in which there may be also present a double bond in the 16,17-position and functional derivatives of such compounds, such as esters, ethers, ketals, acetals and the like.

In those of the aforementioned compounds in which R represents a hydrogen atom the aldehyde group in 17- position may be oxidized by conventional methods illustrated in the said application to a carboxy group, to obtain the etienic acids of the present invention. These etienic acids are thus obtainable by total synthesis. On the other hand they may also be converted into 214 hydroxy pregnene compounds according to known methods. For instance their halides are reacted with diazomethane and the diazoketones obtained are converted either direct or by way of the 2l-halides into 21-hydroxy pregnene compounds. The acid halides can also be reacted with methyl metal compounds, for example those of magnesium, cadmium or zinc; there may also be mentioned compounds of mercury, copper, aluminum, tin and the alkali metals. There are thus obtained the methyl ketones which can be converted into the hydroxy methyl ketones according to known methods. One of these methods for instance consists in treating the methyl ketone with an oxalic acid ester in the presence of an alkaline condensing agent, reacting the so-obtained oxalo acid ester in the form of an alkaline metal salt with halogen, preferably iodine, splitting the 21-halogen oxalo acid ester with the formation of the 2l-halide and converting the latter in known manner into the 21-hydroxy compound.

The compounds of the present invention may also be obtained from known compounds by synthesis when saturated or unsaturated 3,11-dioxygenated 17- or 20- keto-steriods or 3-oxygenated 13,17-seco-steroid-17,l3- lactones are oxidatively coverted into 3,11-dioxygenated l3,17-seco-steroid-17,l3-lactones, the latter cleaved into 13-carbinols, water is eliminated directly or indirectly, if desired after the preparation of an ll-keto-group, the side chain in c p-unsaturated ketones obtained is degraded, if desired, in known manner to 2 carbon atoms and/or converted into a substituent which reacts with an acetic acid derivative containing an active methylene group, the methyl group at the double bond is oxygenated and the compounds obtained are reacted to form 3,11,18-trioxygenated steroid-17-carboxylic acids or functional derivatives by way of substituted acetic acid derivatives having an active methine group.

The starting materials are oxidatively converted into the corresponding 3,11 dioxygenated-13,17-seco-steroid- 17,13-lactones, for example, by means of hydroperoxides, such as hydrogen peroxide or peracids, for example, performic acid, peracetic acid or perbenzoic acid, or by a microbiological method, for example, by means of microorganisms of the groups Mucorales, such as Cunninghamella, Actinornycetes, such as Streptomycetes, Curvularia, Penicillium, Cylindrocarpon, Gliocladium, Aspergillus types or Fungi Imperfecti. The latter method is also especially suitable for the introduction of oxygen into the ll-position of lactones to be used in the process of the invention or for the degradation of ZO-ketones to lactones. During these and the subsequent oxidations, especially when chemical oxidizing agents are used, reactive double bonds in unsaturated rings of the starting materials, for example, a double bond in the 5,6-position, are protected, if desired, intermediately, for example, by additive combination with halogen or a hydrogen halide or conversion into an i-steroid. Any free hydroxyl groups present are advantageously temporarily protected wholly or in part, for example, by esterification or etherification.

A further step in the process consists in splitting up the lactone so obtained into a 13-carbinol, especially by means of a hydrolyzing agent such as an alkali hydroxide, an alkaline earth metal hydroxide, or aluminum oxide, or with an organo-metal compound such as a Grignard compound, for example methyl-, or phenylrnagnesium halide, or an alkylor aryl-lithium compound. If the treatment is carried out with only about one equivalent of alkyl magnesium halide, it is at first possible only to cause the addition of an alkyl radical without splitting up the ring.

After a different substituent in the ll-position has been converted in known manner into a keto group, the elimination of the 13-hydroxyl group takes place very easily. Normally it takes place directly with elimination of Water, generally with the reagents used for splitting up the lactone ring. In the case of the dihydropyrane derivatives or the corresponding 7-ring compounds which are at first obtained, if desired, with alkyl magnesium halides, water is in effect split off indirectly by the oxidation and splitting oif of the acyloxy group in the 13-position. After the splitting up of the lactone ring by means of at least two equivalents of organo-metal compound, and simultaneously with the splitting off of water in the nucleus or independently thereof, water may be split off in the side chain, for example, by means of an excess of the organo-metal compound or by an agent customarily used for splitting oif water.

A further step which may be carried out if desired, is the conversion of a 16-substituent into a hydroxyl, 0x0 or amino group, especially by means of a hydrolysing agent. The side chain in the ,fi-UIISEIIUI'EItCd ketone is then degraded in a manner in itself known to two carbon atoms, and/or converted into a substituent which reacts. with an acetic acid derivative containing an active methylene group. For the degradation there is advantageously used an oxidizing agent such as ozone, chromic acid or lead tetraacetate, or the silver salt of the carboxylic acid may be degraded by means of halogen by the method of Hunsdiecker.

In order to oxygenate the methyl group at the double bond it may, for example, be hydroxylated or acyloxylated, directly, for example, by means of selenium dioxide, lead tetraacetate, or indirectly, for example, by halogenation, ketone splitting and subsequent reaction, for example, by means of a salt of a carboxylic acid such as an alkali acylate or silver acylate. If it is desired, for example, to bring about oxogenation, this step may be carried out by oxidation of the hydroxylated compound or directly. Thus, for example, the starting compound may be reacted with selenium dioxide or the above mentioned halide may be converted by the method of Krohnke by way of the quaternary ammonium salt and the nitrone. In this case also derivatives of the aldehyde group may be obtained intermediately, for example, acetals with monohydric or polyhydric alcohols, and also diacylates. The aforesaid total conversion of the methyl group at the double bond may also be carried out at a different stage in the synthesis and may be interrupted by other later process steps.

Likewise, at any intermediate stage in the synthesis, a A -3-keto group may be introduced in known manner, especially by halogenation and the splitting off of hydrogen halide, for example, by means of a hydrazine such as semi-carbazide or by means of pyruvic acid, and the oxidation of a 3-hydroxyl group and the halogenation may be carried out in a single reaction by means of an alkyl hydrohalide. The A -3-keto group so produced may, if desired, be subsequently protected intermediately, for example, by ketalization or the formation of an enol derivative by a method in itself known.

An essential step of the process is the conversion to a steroid by way of a substituted acetic acid derivative containing an active methine group, such as an acetoacetic acid derivative or malonic acid derivative. This is carried out, for example, by subjecting a primary 16-halide or pseudo-halide to a reaction of the character of an acetoacetic acid ester or malonic acid ester synthesis and to intramolecular condensation according to the method of Michael, or by first carrying out a Michael condensation and subjecting the resulting acetic acid derivative to ring closure. As is usual in acetoacetic acid ester and malonic acid ester syntheses the resulting derivative containing one or two substituted acid radicals is subjected in known manner to a ketone or acid splitting.

In a stage following ring closure to form the steroid the ll-keto group can reductively be converted into an ll-hydroxyl group, advantageously an llfi-hydroxyl group, for example, by means of a light metal hydride such as an alkali metal boron hydride or alkali metal aluminum hydride.

One form of the process for the preparation of the starting materials is illustrated by the following diagram of formulae AeOOH Ph rt, mpg

lNBS

\/C O OH 2 2 010 III i Na-Malonic ester I "l l l x a O O 0 CH COOH Cg COOR \Cfi COOR 000 0 COOR -00i KOC(CH l:O l:0 O\ O [O XXIV XXIII XXII lNaBHt l' l "I a x a O CH COOH Cg COOH HO R10, Py O O R=F CCO XXV XXVI The resulting compound XXV, on ketal and acetal is dissolved in benzene, and the solution is chromato- CH OCO coon coon no- I U tfj C This compound may be converted into the 18,11-lactone of A -3,2O-diox0-115,21-dihydroxy-pregnen-1-8 acid by the methods mentioned above and the so obtained lactone after ketalization in 3- and 20-position is converted into aldosterone according to the process of copending application Serial No. 770,006, now U.S. Patent No. 3,014,904, filed October 28, 1958 by Tadeous Reichstein et al.

The compounds of the present invention thus are obtainable by synthesis starting from known compounds and are convertible into 18-oxygenate-d A -3,20-dioxo 11fl,21- dihydroxy-pregnene compounds, among which there are important new hormones of the adrenal glands, such as aldosterone and its derivatives and related compounds easily convertible into such compounds. They are therefore intermediates for the preparation of physiologically important products.

The following Preparations relate to the production of the starting materials for the compounds of the present invention:

PREPARATION 1 A solution of 0.693 gram of 3a-acetoxy-11,17-diketotestane (I) and 0.035 gram of paratoluene sulfonic acid in 14 cc. of glacial acetic acid resisting to chromic acid is mixed with 2.27 cc. of a LOG-molar solution of acetoperacid in glacial acetic acid, and the mixture is maintained in the dark for 72 hours at room temperature. A large quantity of water is then added, the partially prepicipated reaction product is taken'up in methylene chloride, the extract is washed with an 0.5 N-solution of sodium hydrogen carbonate and water, dried with sodium sulfate and evaporated. The pale yellow residue graphed by the fractional elution method over a column of 21 grams of silica gel prepared with benzene. As elutriating agents there are used in succession benzene, ether and ethyl acetate. At first a small amount of an amorphous substance is dissolved out with benzene. From the succeeding ether and ethyl acetate elutriates there is obtained by crystallisation from a mixture of benzene and hexane pure 3a-acetoxy-1l-keto-13a-hydroxy-13,17-secotestane-l7-acid-17,13-lactone of the Formula II melting at 178-180 C.

The same compound can also be obtained by treating 3a-acetoxyor 3a-hydroxy-11,20-diketo-pregnane with an aerated and stirred submerged culture of Aspergz'llus flavus, Penicillium radametzi, Penicillium lilacinum or Penicillium chrysogenum, Streptomyce lavendulate, F usarium salami or caucasicum, and acylating the reaction product with acetic anhydride in pyridine.

PREPARATION 2 0.732 gram of 3u-acetoxy-1l-keto-13a-hydroxy-13,17- seoo-testane-17-acid-17,13-lactone, dissolved in 7.5 cc. of benzene, is run in the course of 30 minutes into a boiling solution of phenyl-magnesium bromide prepared from 3.21 grams of magnesium and 12.6 cc. of bromobenzene in cc. of ether. The whole is boiled for 22 hours, then 7.5 cc. of a 4-molar aqueous solution of ammonium chloride is added while cooling with ice, the benzene and the resulting diphenyl are distilled with steam. After cooling the mixture, the precipitated crude product is taken up in ether, the ethereal extract is washed with a 2-molar solution of ammonium chloride and water, dried with sodium sulfate and evaporated.

In order to split off water the crude carbinol so obtained is covered with 12 cc. of glacial acetic acid and the mixture is boiled for 16 hours with the exclusion of moisture. After cooling the mixture, water is cautiously added and the precipitated reaction product is filtered off with suction after allowing the mixture to stand for several hours. It is thoroughly washed with water and dried. By recrystallizing it form methanol and a mixture of ethyl acetate and isopropyl ether there is obtained pure A -1B-(3,3' diphenyl allyl) 7a-acetoxy-4-keto- 2,4b 3-dimethyl 4aa,8a,8,10a-,8 dodecahydro phenanthrene of the formula IV. In the ultra-violet spectrum the compound exhibits a maximum=244 11111.; log 5:4.52.

1 1 PREPARATION 3 To a solution of 0.965 gram of A -1fi-(3',3'-diphenyl allyl) 7a acetoxy-4-keto-2,4b5-dimethyl-4aa,8a[3,lOafidodecahydro-phenanthrene in 11.2 cc. of ethylene chloride and 24.0 cc. of acetic acid of 83.3 percent strength there is added at 3 C. while stirring in the course of 4 hours a solution of 0.480 gram of chromium trioxide in 4.32 cc. of acetic acid of 83.3 percent strength. After the mixture has been stirred for 18 hours while cooling with ice, there are added 7.5 cc. of an 0.1-molar solution of sodium hydrogen sulfite, and the organic solvent is distilled 01f in vacuo while replacing the evaporated water. The residue is extracted by agitation with a mixture of ether and methylene chloride (3:1), the extract is first washed with water, 0.5N-sulfuric acid and water, and then the acid reaction products are exhaustively extracted with a 2 N-solution of sodium carbonate. The combined alkaline extracts are acidified with 2 N-hydrochloric acid and extracted by agitation with a mixture of ether and methylene chloride (3:1). The ether-methylene chloride extract is washed with water and dried with sodium sulfate, and then mixed with an 0.4l-molar solution of diazo methane in ether until the yellow coloration persists, and the extract is evaporated after 20 minutes. i

The crude ester so obtained is dissolved in 3.0 cc. of anhydrous pyridine and mixed with 2.0 cc. of acetic anhydride. After 16 hours the mixture is evaporated in vacuo with the repeated addition of toluene, and the residue is recrystallized from a small quantity of methanol. There is obtained in the form of colorless crystals A 7a acetoxy-4-keto-2,4b,B-dimethyl-4aa,8a/8,10aB-dodecahydro-phenanthryl-(1fi)-acetic acid methyl ester of the Formula VI, which exhibits in the ultraviolet absorption spectrum a A max=240 m,u, log 6=4.20. The acid V can also be obtained directly from the 3a-acetoxy-11- keto 13oz hydroxy-13,l7-seco-testane-17-acid-17,13-1actone of the Formula II described in Preparation 1. 20 mg. of lactone II are dissolved in benzene and allowed to stand for 4 hours in a column of 1 gram of (neutral) alumina. It is then washed with benzene, ether and chloroform, practically no material being separated. Washing with a mixture of methanol and glacial acetic acid then elutriates the A 7a acetoxy-4-keto-2,4bB-dimethyl 4aa,8a 8,10,B dodecahydro-phenanthryl-(113)- acetic acid of the Formula V.

PREPARATION 4 1.450 gram of A -7ot-acetoxy-4-keto-2,4bfi-dimethy1- 4aa,8a{3,10a,8 dodecahydrophenanthryl-(1,6)-acetic acid methyl ester and 0.890 gram of N-bromo-succinimide are covered with 30 cc. of carbon tetrachloride, and the mixture is boiled for 20 minutes under reflux while being strongly illuminated. After cooling the mixture in ice water the succinimide is filtered off with suction and the pale yellow filtrate is evaporated in vacuo. The residue yields by recrystallization from a mixture of ether and isopropyl ester A -7a-acetoxy-4-keto-2-(bromomethyl)-4b[3- methyl 4aa,8afi,l0afi dodecahydrophenanthryl-(1,8)- acetic acid methyl ester of the Formula VII.

A solution of 0.883 gram of the crystalline bromide in 40 cc. of methanol is mixed with 7.6 cc. of a 2.63 N-methanolic solution of hydrobromic acid, and the whole is allowed to stand for 22 hours. After dilution with a large quantity of water the mixture is extracted by agitation with a mixture of ether and methylene chloride (3:1), the extract is washed with water, 0.5 N-solution of sodium acetate and water, then dried with sodium sul fate and evaporated. The residue is crude A -7a-hydroxy- 4 keto 2-(bromomethyl)-4b,8-methyl-4aa,8a,8,lOafi-dodecahydro-phenanthryl)-(1fl)-acetic acid methyl ester of the Formula VIII. It is advantageously further worked up directly.

The free 7a-hydroxy-con1pound so obtained is dissolved in 10 cc. of anhydrous acetone, 0.982 gram of anhydrous potassium acetate is added, and the mixture is boiled for 6 hours with the exclusion of moisture. After cooling the mixture, 20 cc. of water are added and the acetone is distilled off in vacuo. The precipitated resin is taken up in ether, and the extract is washed with water, an ice cold 0.1 N-solution of sodium hydrogen carbonate and water, dried with sodium sulfate and evaporated. For the purpose of purification the residue is dissolved in an mixture of hexane and benzene (9:1) and chromatographed by the fractional elution method over a column of 25 grams of aluminum oxide prepared with hexane. As elutriating agents there are used a mixture of hexane and benzene (9:1) and (1:1), benzene, a mixture of benzene and ether (9:1) and ether. The last portions eluted with benzene and also the fractions subsequently obtained with benzene-ether (9:1) and ether crystallize from a mixture of ether and petroleum ether. By recrystallization from the same pair of solvents there is obtained pure A -7ot-hydroxy-4-keto-2-(acetoxy-methyl) 4bB-methyl-4aa,8afl,10afl-dodecahydro-phenanthryl- (IQ-acetic acid methyl ester of the Formula IX.

PREPARATION 5 1.514 gram of A -7u-hydroxy-4-keto-2-(acetoxy-methyl) 4bfi-methyl-4aa,8a,B,10aB-dodecahydro-phenanthryl- (113)-acetic acid methyl ester are added while rinsing with 2.2 cc. of pyridine to a suspension of the chromic acidpyridine complex prepared by introducing 0.440 gram of chromium trioxide into 8.8 cc. of pyridine. The mixture is stirred for 18 hours at room temperature. A large quantity of water is then added, the mixture is extracted with ether by agitation, the extract is washed in succession with water, ice cold 0.5 N-sulfuric acid and water, and then dried with sodium sulfate and evaporated. The residue is crystallized from a mixture of ether and isopropylether. The resulting A -4,7-diketo-2-(acetoxy-methyl)Abe-methyl 4aa,8aa,10a,8 dodecahydro-phenanthryl-(1B)-acetic acid methyl ester of the Formula X exhibits, in contradistinction to the starting material, no hydroxyl band at 2.7;]. in the infra-red absorption spectrum.

0.753 gram of the above diketone is dissolved in 15 cc. of glacial acetic acid, 0.30 cc. of a l-molar solution of hydrogen bromide in glacial acetic acid is added, and 4.2 cc. of a solution of bromine and sodium acetate in glacial acetic acid, which is 0.5-molar with respect to both reagents, is slowly added dropwise while stirring. When the bromine is practically completely consumed, cc. of water are added and the mixture is extracted by agitation with benzene. The extract is washed in succession with water, 0.5 N-sodium hydrogen carbonate and water, dried with sodium sulfate and evaporated in vacuo. The residue which slowly crystallizes on standing is crude A -4,7- diketo 2 (acetoxy-methyl)-8a-bromo-4bfi-methyl-4aa, 8a/3,10a/i-dodecahydro-phenanthryl-(1B)-acetic acid methyl ester of the Formula XI, and is purified by recrystallization from methanol and a mixture of ether and isopropylether.

To 0455 gram of the above bromide in 75 cc. of ethanol of percent strength is added, while stirring, 0.300 gram of semicarbazide and 0.75 cc. of glacial acetic acid. After 16 hours 75 cc. of water are cautiously added, the precipitated semicarbazone is collected on a suction filter after allowing the mixture to stand for one hour, and the filter residue is washed with water and dried in vacuo. For the purpose of splitting it is covered with 15 cc. of glacial acetic acid and 5.0 cc. of an aqueous 0.4 N-solution of pyroracemic acid and stirred for 38 hours in an atmosphere of carbon dioxide. After the addition of 20 cc. of water the mixture is extracted by agitation with a mixture of ether and methylene chloride (3:1) the extract is Washed in succession with 2 N-solution of sodium hydrogen carbonate, water, 1 N-hydrochloric acid and water, and then dried with sodium sulfate and evaporated. The residue is dissolved in a mixture of hexane and benzene and chromatographed over a column of 20 grams of '13 aluminum oxide prepared with hexane. Elutriation is carried out in succession with a mixture of hexane and benzene (9:1) and (1 :1), benzene, a mixture of benzene and ether (9:1) and ether, and the fractions dissolved are tested by paper chromatography. The practically unitary benzene elutriates and benzene-ether elutriates crystallize from methanol. In this manner there are obtained practically colorless crystals of A -4,7-diketo-2-(acetoxymethyl) 4b,8 methyl-4am,10afi-decahydro-phenanthryl- (MD-acetic acid methyl ester of the Formula XII which exhibits in the ultraviolet absorption spectrum a A max=246 m log 4.53.

PREPARATION 6 A mixture of 1.498 gram of A -4,7-diketo-2-(acetoxymethyl) 4bfi methyl-4am,10afi-decahydro-phenanthryl- (1,B)-acetic acid methyl ester, 2.25 cc. of ethylene glycol, 30 cc. of ethylene chloride and 0.016 gram of para-toluene sulfonic acid is subjected to slow distillation for 3 hours during which the moist ethylene chloride which distils off is replaced continuously by introducing fresh solvent. After being cooled, the reaction mixture is extracted in succession with a 1 N-solution of potassium carbonate and water, dried with sodium sulfate and evaporated. The resulting A 4-keto-2-(hydroxy-methyl)-7-ethylene-dioxy 4b;3 methyl-4aa,10afi-decahydro-phenanthryl-(1,8)- acetic acid methyl ester of the Formula XIII is purified by recrystallization from a mixture of ether and petroleum ether.

0.753 gram of the above ketal is dissolved in 20 cc. of anhydrous tetrahydrofurane, a solution of 0.950 gram of lithium aluminum hydride in 13.5 cc. of anhydrous tetrahydrofurane is added dropwi-se, while stirring, and stirring is continued for 36 hours at room temperature. 2.7 cc. of water are then added dropwise While cooling externally with ice Water, the mixture is freed from precipitated inorganic material, the filter residue being washed with tetrahydrofurane, and the filtrate is evaporated in vacuo. The residue is crude A 4fl-hydroxy-lfi-(2'-hydroxy-ethyl)- 2 (hydroxy methyl)-7-ethylene-dioxy-4bfi-methyl-4aa, 10aB-decahydro-phenanthrene f the Formula XIV.

The crude triol so obtained is allowed to stand with 3.0 cc. of acetic anhydride in 4.5 cc. of pyridine for 16 hours at room temperature. The reaction mixture is then evaporated in vacuo with the repeated addition of toluene, the residue is dissolved in benzene, and the solution is filtered through a short column of active carbon. On evaporation the clear filtrate leaves behind a pale yellow residue. By recrystallization from methanol there is obtained A -4fi-hydroxy1,8-(2'-acetoxy-ethyl) -2-(acetoxymethyl)-7-ethylene-dioxy-4bB-methyl-4au,10a,8 decahydro-phenanthrene of the Formula XV.

PREPARATION 7 A solution of 1.738 grams of A -4fi-hydroxy-1fl-(Z- acetoxy ethyl) 2 (acetoxy-methyl)-7-ethylene-dioxy- 4bB-methyl-4aa,10aB-decahydro-phenanthrene in 13.2 cc. of pyridine is added to 0.440 gram of chromium trioxide in 8.8 cc. of pyridine, and the mixture is stirred for 62 hours at room temperature. The whole is then diluted with a large amount of water, extracted by agitation with a mixture of benzene and ether (1:2), the extract is filtered through Super Cel (reg. trademark), then washed with water, dried with sodium sulfate, and evapo rated in vacuo. The residue is dissolved in a mixture of hexane and benzene, (3:1), and chromatographed by the fractional elution method over a column of 52 grams of aluminum oxide prepared with hexane. Elutriation is carried out in succession with a mixture of hexane and benzene (3:1), benzene, a mixture of benzene and ether (9.11) and (1:1), ether and a mixture of ether and ethyl acetate (9: 1). The fractions are tested by paper chromatography. The practically unitary elutriates which exhibit a strong absorption in the ultraviolet contain A -4- keto-1[5-(2'-acetoxy-ethyl)-2-(acetoxy-methyl) 7 ethyl- 14% ene-dioxy-4bB-methyl-4aa,Nae-decahydro phenantrene of the Formula XVI.

To 0.865 gram of the above ketone in 20 cc. of benzene are added 20 cc. of a 1 N-methanolic solution of potassium hydroxide, and the whole is heated at the boil for 2 hours. After cooling the mixture, 10 cc. of water are added, carbon dioxide is first introduced in order to neutralize the potassium hydroxide, and then the methanol is distilled off until the final volume of the mixture is small. After the addition of water, the crude product is taken up in a mixture of ether and ethyl acetate (1 1), and the extract is Washed with water, dried with sodium sulfate and evaporated in vacuo. The residue consists substantially of A 4 keto-lfi-(2'-hydroxy-ethyl)-2-hydroxymethyl)-7-ethylene-dioxy-4b5-methyl-4aa,10afl decahydro-phenanthrene of the Formula XVII, and is then further worked up immediately.

The crude diol so obtained is dissolved in 4 cc. of pyridine, 1.41 grams of para-tosylbromide are added while stirring, the mixture is initially cooled for one hour to about 10 C., and then allowed to stand for 72 hours at room temperature While stirring occasionally. At the end of this period the greater part of the pyridine is evaporated cautiously in vacuo, the crystals are stirred with a mixture of methanol and ether (1:1) and the mixture is filtered with suction. The resulting N-{[A 4 keto-1 8-(2'- tosyloxy-ethyl)-7-ethylene-dioxy-4bB methyl 4aa,10afidecahydrophenanthryl-(Z) ]-methyl}-pyridinium salt of the Formula XVIII is purified by recrystallization from a mixture of methanol and acetone.

PREPARATION 8 10 cc. of an 0.2 N-solution of sodium hydroxide are added to a solution of 1.289 grams of N-{[A -4-keto-1B- (2' tosyloxy ethyl) 7 ethylene-dioxy-4bfi-methyl-4aa, IOaB decahydro phenanthryl-(2)]-methyl}-pyridinium bromide and 0.330 gram of para-nitrodimethylaniline in 30 cc. of methanol and 20 cc. of water. After allowing the mixture to stand for 48 hours, it is filtered with suction, the filter residue is washed with methanol of 50 percent strength and water, and the resulting [A -4-keto-1fl- (2'-tosyloxy-ethyl)-7-ethylene-dioxy 4hr? methyl 4am, 10aB-decahydro-phenanthryl-(2)] N (para dimethylamino-phenyl)-nitrone of the Formula XIX is crystallized from methanol with the use of methylene chloride as solution promoter.

0.635 gram of the nitrone is agitated with 15 cc. of 1 N-hydrochloric acid and 60 cc. of a mixture of ether and benzene (3:1) for one hour in an atmosphere of hydrogen. The organic phase is then washed with water, 0.5 N-sodium hydrogen carbonate solution and water, dried with sodium sulfate and evaporated in vacuo. The residue consists of crude A -4,7-diketo-1e-(2-tosyloxyethyl)-2-formyl-4bfi-methyl-4aa,10a,6 decahydro phenanthrene of the Formula XX.

The resulting crude aldehyde is dissolved in 25 cc. of ethylene chloride, 2.5 cc. of ethylene glycol and 0.019 gram of para-toluene sulfonic acid are added, and the mixture is subjected to slow distillation for 3 hours. The moist ethylene chloride which passes over is continuously replaced by fresh ethylene chloride. After being cooled, the solution is agitated with an 0.5 N-solution of potassium carbonate and water, dried with sodium sulfate and evaporated. The residue is dissolved in a mixture of hexane and benzene (1: 1), and for the purpose of purification the solution is chromatographed by the fractional elution method over a column of 32 grams of aluminum oxide prepared with hexane. Elutriation is carried out in succession with a mixture of hexane and benzene (1:1) and (1:3), benzene, and a mixture of benzene and ether (9:1). The benzene fractions and benzene-ether fractions, which are practically unitary according to tests by paper chromatography, are crystallized from ether, and there is obtained A -4-keto-1fl-(2' tosyloxy-ethyl) 2 (ethylene- 1 dioxy-mcthyl)-7-ethylene-dioxy 4bfl methyl 4aot,10a5- decahydro-phenanthrene of the Formula XXI.

The following examples illustrate the invention:

Example 1 2.720 grams of A -4-keto-1fi-(2-tosyloxy-ethyl)-2- (ethylene-dioxy-methyl)-7-ethylene dioxy 4bfl methyl- 4aa,10a 8-decahydro-phenanthrene and 5 cc. of absolute ethanol are added to a solution of sodium malonic ester, prepared from 0.130 gram of sodium, cc. of absolute ethanol and 0.880 gram of malonic acid diethyl ester, and the mixture is heated at the boil for 8 hours in an atmosphere of nitrogen. Water is added to the cooled mixture, the greater part of the ethanol is distilled off in vacuo, and the crude alkylation product is taken up in a mixture of ether and benzene (1:1). The solution is washed with water, dried with sodium sulfate and evaporated.

To a solution of the residue in 100 cc. of tertiary butanol there are added 5 cc. of an 0.1-molar solution of potassium tertiary butylate in tertiary butanol, and the whole is heated on a water bath for 2 hours in an atmosphere of nitrogen. The solvent is then distilled off in vacuo while introducing water, the precipitated condensation product is taken up in benzene, the solution is washed with water, dried with sodium sulfate and evaporated. The residue is dissolved in benzene and the solution is chromatographed by the fractional elution method over a column of 75 grams of aluminum oxide prepared with benzene. Elutriation is carried out with benzene, a mixture of benzene and ether (9:1), ether, and a mixture of ether and ethyl acetate (9:1). The fractions dissolved out with benzene-ether (9: 1) and ether, which are unitary according to tests by paper chromatography and exhibit no absorption in the ultra-violet, yield when recrystallized from a mixture of ethyl acetate and ether A -3,18-bisethylene-dioxy-l 1-keto-androstene-17,17-dicarboxylic acid diethyl ester of the Formula XXIII (R=Et).

1.065 grams of the dicarboxylic acid ester are saponified by being boiled for 2 hours in an atmosphere of nitrogen with cc. of a 4 N-methanolic solution of potassium hydroxide. After the addition of water, neutral substances which are still present are removed from the mixture by agitating it with a mixture of ether and benzene (1:1), and then 0.1 N-sulfuric acid is added, while cooling with ice and stirring, until the reaction is neutral to litmus. The precipitated free dicarboxylic acid is then taken up in a mixture of ethyl acetate and benzene (3:1), the solution is washed with 0.01 N-ammonium carbonate solution and water, dried with sodium sulfate and evaporated. The residue is heated with 25 cc. of ethylene glycol at 120125 C. until carbon dioxide ceases to be split off, the solution is then cooled and water is added cautiously. The precipitated 13 -3,18-bis-ethylene-dioxyll-keto-etienic acid of the Formula XXIV is filtered off after allowing the mixture to stand for several hours, and the filter residue is washed with water and dried in vacuo.

Example 2 A mixture of 0.865 gram of A -3,l8-bis-ethylene-dioxyll-keto-etienic acid, 0.756 gram of sodium boron hydride and 40 cc. absolute methanol is boiled under reflux for 2 hours. After cooling the mixture in ice water it is neutralized, while stirring, by first adding about 10 cc. of 2 N-acetic acid and then water until the separation of the reduction product is complete. By filtering, washing the filter residue with water and drying there is obtained A -3,l8-bis-ethylene-dioxy-1lfl-hydroxy-etienic acid of the Formula XXV.

2 cc. of trifiuoracetic anhydride are added at 0 C. to a solution of 0.435 gram of the hydroxy-acid in 5 cc. of pyridine, and the mixture is allowed to stand at room temperature for 4 hours. It is then mixed with 5 cc. of dioxane and 2 cc, of water, and the Whole is allowed to stand for a further 2 hours. It is then extracted by agitation with a mixture of ether and chloroform (3 :1), the extract solution is washed with water, dried with sodium sulfate and evaporated in vacuo with the repeated addition of benzene. The residue is crude A -3,18-bis-ethylene-dioxy- 11fi-trifluoracetoxyetienic acid of the Formula XXVI.

Example 3 4.597 mg. of aldosterone hydrate are dissolved in 0.7 cc. of purest methanol and treated with 0.9 cc. of sodium periodate solution. (This solution is prepared as follows: 1.25 mg. of Na H IO are suspended in about cc. of water and treated with shaking with 2 N sulfuric acid until the reaction is distinctly acid to litmus, whereupon the whole is made up with water to cc.) After one hour long needles are formed. After standing for two hours, the methanol is evaporated under vacuum and the residue extracted by shaking first with 1 cc. of chloroform, then with 7 cc. of chloroform-ether (1:3) and finally twice, in each case with 6 cc. of chloroform-ether 1:3). The organic solvents are then washed at about 0 C. twice with 0.6 cc. of sodium carbonate solution and twice with 0.6 cc. of Water, then dried over sodium sulfate and evaporated under vacuum. The crystalline neutral portion thus obtained amounts to 3.17 mg. and constitutes the 20,18-lactone of the 18,11-cyclo-semi-acetal of A -3,18- dioxo-l1,8-hydroxy-etienic acid of the formula After recrystallization from acetone-ether it is obtained in the form of colorless pointed prisms or leaflets of M.P. 307313 C. (with brown coloration);

- 2 (c.=0.9154 in chloroform). By concentration of the mother liquor, a further quantity of the above lactone can be obtained, which after sublimation under 0.03 mm. pressure and at 192210 C. melts at 307-313 C.

For the isolation of the acid formed in the oxidation, the aqueous sodium carbonate solutions obtained as above are treated at 0 C. with 1.4 cc. of 2 N-sulfuric acid until the whole is just acid to Congo red and then extraction carried out three times by shaking with 7 cc. of chloroform-ether (1:3) each time. The product is then in turn washed three times with 0.5 cc. of water each time, dried over sodium sulfate and the solvent evaporated. The crystalline residue amounts to 0.45 mg. and constitutes the crude A -3,18-dioxo-1lfi-hydroxy-etienic acid of the formula:

This compound yields, on recrystallization from acetoneether, short colorless prisms which on heating under the microscope are converted at about 220240 C. into transparent leaflets which melt at about 295303 C. with brown coloration.

Detection of the formaldehyde formed in the above reaction: The sulfuric acid aqueous solutions obtained above are adjusted with 0.2 cc. of 2 N-sodium carbonate solution to a pH of about -6 and then evaporated to complete dryness under 80 mm. pressure and at 60 C. The distillate is treated with 5 mg. of dimedone and 0.5 cc. of glacial acetic acid, heated for one hour to 100 C. and left to stand for 16 hours at about 18 C. The separated crystals, after filtration with suction and Washing with Water, amount to 0.66 mg, melt at 192-195 C. and constitute the formaldehyde-dimedone derivative. From the acetic acid aqueous solution, by extraction with ether, a further quantity can be isolated.

Example 4 0.4 mg. of the A -3,18-dioxo-1lfi-hydroxy-etienic acid described in Example 3 are sublimed in a small flask under 0.03 mm. pressure and at 210-230 C. The sublimate is dissolved in 2.5 cc. of chloroform and 7.5 cc. of ether and washed with 0.5 cc. of 2 N-sodium carbonate solution, 0.5 cc. of water, 0.3 cc. of 2 N-sodium carbonate solution and then three times with 0.3 cc. of water each time. These solutions are further treated in a second separating funnel with 4 cc. of chloroform-ether. The chloroform-ether solutions are combined, dried over sodium sulfate and give after evaporation 0.3 mg. of neutral residue which constitutes the 20,18-lactone of the 18,11- semiacetal of the A -3,18-dioxo-1lfl-hydroxy-etienic acid described in Example 3.

Example 5 9.692 mg. of the 20,18-lactone of the 18,1l-cyclosemiacetal of the A -3,18-dioxo-1lfi-hydroxy-etienic acid, described in Examples 3 and 4, are hydrogenated in glacial acetic acid solution in the presence of 5 mg. of prehydrogenated platinum oxide. After 7 minutes 1.235 cc. (calculated quantity 1.32 cc.) of hydrogen have been taken up and the hydrogenation is interrupted. The hydrogenation product (10.2 mg.) obtained after working up, consists for the most part of colorless leaflets of M.P. 222- 255 C. (with decomposition) and constitutes a mixture of the 20,18-lactones, isomeric in 5-position, of the 18,11- cyclo-semiacetals of the 3,11 dihydroxy-18-oxo-etianic acids of the formula:

By treatment with a mixture of acetic anhydride and pyridine there can be isolated the 20,18-lactone of the 18,11-cyclo-semiacetal of 3,8-acetoxy-11fi-hydroxy-18-oxoallo-etianic acid which crystallizes from methanol in the form of flat needles melting at 218225 C.; [a] =0 (oz-0.5246 in chloroform).

The non-acetylated mixture can be converted into the known 3,11-diketo-allo-etianic acid methyl ester in the following manner:

The above hydrogenation product (10.2 mg.) is heated in a mixture consisting of 0.03 cc. of hydrazine hydrate and a solution of 12 mg. of sodium in 0.5 cc. of ethanol in an evacuated bomb tube for 13.5 hours to 148 C. After working up there are obtained 3 mg. of neutral portion and 7.8 mg. of acid constituents. The latter, after crystallization from acetone-ether, yield 3.6 mg. of 18,11-

18 lactone of 35,11p dihydroxy-allo-etianic acid-18-acid of the formula which melts at 270-275 C. (with decomposition). From the mother liquor of the acid there is obtained a further 1.1 mg. of substance of M.P. 252268 C. (with decomposition). The residue of the acid fraction (3.2 mg.) is amorphous.

The amorphous acid (3.2 mg.) is esterified with diazomethane and the methyl ester thus obtained is oxidized in glacial acetic acid solution with 2 mg. of chromium trioxide. After two hours, the oxidation is interrupted, the reaction solution worked up and the crude product chromatographed over 300 mg. of aluminum oxide. From the crystallized fractions, after recrystallization from ether-pentane, crystals are obtained of M.P. 205212 C., which are identical with known 3,11-dioxo-allo-etianic methyl ester.

From' the neutral portion, the 20,18-lactone of 35,11 8, 18-trihydroxy-allo-etianic acid of the formula:

can be isolated.

Example 6 The 18,11-1actone of the 3,8-1lfl-dihydroxy-alloetianic acid-18-acid (4.7 mg.) obtained according to Example 5 is esterified with diazomethane and the crude product recrystallized from acetone-ether. The resulting 18,11-lactone of 33,1l,B-dihydroxy-allo-etianic acid methyl ester- 18-acid of formula o-oo 000011,

melts at 232236 C.; [cc] +8l.5 :t6" (c.:0.362 in chloroform).

3.5 mg. of the above lactone-methyl-ester are dissolved in 0.4 cc. of purest glacial acetic acid and treated 19 lactone of 3-ox0-11 8-hydroxy-allo-etianic acid methyl ester-18-acid of the formula OCO COOCH The above 3/3-hydroxy-lactonemethyl ester on acetylation with acetic anhydride-pyridine yields the 18,11- lactone of 3,8-acetoxy-1lfl-hydroxy-allo-etianic acid methyl ester-18-acid. It crystallizes fro ma mixture of methanol and acetone in the form of rhombic leaflets melting at 194-195 C.;[cz] =-|64.1 (c.:0.4836 in chloroform).

Example 7 1.267 mg. of aldosterone monoacetate of M.P. 190- 192 C. are dissolved in 0.12 cc. of glacial acetic acid and treated with 0.01 cc. of 2% chromium trioxide-glacial acetic acid solution. After minutes the chromium trioxide is consumed and a further 0.01 cc. of the same solution is added. After 3 /2 hours a little methanol is added to the reaction solution and the whole allowed to stand for a further hour and then evaporated under vacuum at C. The residue is taken up in chloroform-ether (1:3), washed at 0 C. twice with 0.15 cc. of N-sodium carbonate solution each time and twice with 0.1 cc. of water each time, the chloroform-ether solution dried and evaporated under vacuum. The neutral portion is recrystallized twice from acetone-ether and washed with ether and pentane. The melting point of the resulting 18,11- lactone of A -3,20-dioxo-11/8-hydroxy-2l-acetoxy-pregnen-18-acid of the formula CHQOAQ which occurs in colorless leaflets (also in part in clusters), is 187190 C.

After further crystallization from a mixture of chloro form and ether this compound melts at 198-199 C. (flat needles); [a] =+ll7.2 (c.=0.6186 in chloroform).

For hydrolysis of the acetate group, 6,3 mg. of the above compound are dissolved in 1 cc. of methanol, a solution of 7 mg. of potassium bicarbonate in 0.24 cc. of water are added and the whole is allowed to stand at 18 C. for 48 hours. After acidification with dilute hydrochloric acid it is extracted with a mixture of chloroform and ether (1:3), the extract washed first with water, then with sodium bicarbonate solution and again with water, dried, and the solvent evaporated under reduced pressure. The neutral portion so obtained amounts to 3.5 mg. and is the 18,11-lactone of A -3,20-dioxo-11 8,21-dihydroxy-pregnen-lS-acid. After recrystallization from a mixture of acetone and ether it forms small grains which melt at 2032l8 C.

For oxidatively splitting the ketol group, 5.8 mg. of this last-mentioned compound are dissolved in 0.6 cc. of methanol, mixed with 1.2 cc. of a sodium periodate solution prepared according to Example 3, and allowed to stand at 20 C. for 1% hours. The reaction mixture is then rendered slightly acid to Congo and extracted with a mixture of chloroform and ether (1:3). After washing the extract with dilute sodium carbonate solution and water, drying, and evaporating the solvent, there are obtained 3 mg. of a neutral portion. To obtain the acid formed in the oxidation, the sodium carbonate solutions are acidified and extracted with a mixture of chloroform and ether (1:3). The chloroform-ether solution, after being washed with water, yields on drying and evaporation 2.8 mg. of the 18,11-lactone of the A -3-oxo-11fihydroxy-ethienic acid-18-acid of the formula OCO COOH which is obtained in the form of fine grains melting at 310320 C. after crystallization from a mixture of acetone and ether. By methylation by means of diazomethane and recrystallization from a mixture of acetone and ether there is obtained the corresponding methyl ester of melting point 219-225 C. (prisms).

The above mentioned acid melting at 310320 C. can be hydrogenated according to the directions given in Example 5. By this means there is obtained the 18,11-lactone of 313,11fi-dihydroxy-allo-etianic acid-18-acid of M.P. 270-275 C. (with decomposition), described in Example 5.

1.7 mg. of the above-described 18,11-lactone of the A -3-oxo-1lfl-hydroxy-etienic acid methyl ester-18-acid of melting point 219-225 C. are dissolved in 1.5 cc. of glacial acetic acid and hydrogenated at 22 C. in the presence of 1.5 mg. of platinum oxide. When the hydrogenation ceases the catalyst is removed by filtration, the filtrate evaporated under reduced pressure and the residue taken up in a mixture of chloroform and ether 1:3). The chloroform-ether solution is then washed twice with water, dried and evaporated in vacuo. The crude residue amounts to 1.7 mg. and melts at 210-230 C. after recrystallization from a mixture of acetone and ether. On further recrystallizing, the melting point rises to 232- 236 C. This compound is identical with the 18,11-lactone of 3,8,11fi-dihydroxy-allo-etianic acid methyl ester- 18-acid described in Example 6.

Example 8 7 mg. of aldosterone hydrate are dissolved in 0.7 cc. of methanol and treated with 1.28 cc. of sodium periodate solution (prepared according to Example 3). After only a few minutes, the separation of crystal needles commences. After 2 hours the methanol is removed under vacuum at 20 C. Then at 0 C., with 2 N H SO a just distinctly acid reaction to Congo red is established and the whole extracted by shaking three times, in each case with 10 cc. of chloroform-ether (1:3). (For the Working up of the aqueous phase see below.)

The three extracts are washed in turn by means of the following solutions: 1 cc. of water, 1 cc. of 0.2 N sodium carbonate, 0.5 cc. of water, 1 cc. of sodium carbonate solution and twice with 0.5 cc. of water each time. The solutions, dried over sodium sulfate, give on evaporation 6.7 mg. of neutral constituents. The combined water and sodium carbonate extracts are acidified with sulfuric acid and extracted by shaking with chloroform-ether (1:3). (For the working up of the acid aqueous phase see below.) The extracts washed with a little water and dried over sodium sulfate give on evaporation 0.4 mg. of A -3,18- dioxo-llfi-hydroxy-etienic acid (see Example 3).

The 6.7 mg. of neutral constituents give from acetoneether 4.5 mg. of the 20,18-lactone, described in Example 3, of the 18,11-cyclosemiacetal of A -3,18-dioxo-11,8-hydroxy-etienic acid of M.P. 309-315 C. (with decomposition). The mother liquors, after distillation in a small ,(total therefore 1.65 mg. CrO =5.0 equivalents).

21 flask or after chromatography over alumina, give a further quantity of the same compound.

Example 9 69 mg. of crude aldosterone-hydrate are dissovled in 6 cc. of methanol and allowed to stand for 1 /2 hours with 12 cc. of sodium periodate solution. Working up by a method analogous to the directions of Examples 3 and 8 gives 21 mg. of the A -3,18-dioxo-1lfl-hydroxyetienic acid described in Example 3 and 37.8 mg. of a neutral fraction. The latter gives from acetone-ether 4.7 mg. of crude 20,18-lactone of the 18,1l-cyclosemiacetal of A -3,l8-dioxo-1lfl-hydroxy-etienic acid of M.P. 285305 C. (with decomposition).

In an analogous manner there are obtained from 19 5 mg. of crude aldosterone-hydrate 95 mg. of the A 318- dioxo-llfi-hydroxy-etienic acid described in Example 3 and 76 mg. of a neutral fraction. The latter yields from acetone-ether 15 mg. of the above described lactone of M.P. 303312 C. (with decomposition).

The mother liquors from the lactone obtained in these two cases are distilled in a small flask under 0.02 mm. pressure. The fractions passing over up to 195 C. bath temperature are discarded. The distillate obtained from 195-230 C. gives from acetone-ether a further quantity of about 0.7 mg. of crystalline lactone of M.P. 275295 C. Chromatography of the remaining mother liquors over alumina (alkali free) including elution with petroleum ether-benzene and pure benzene, gives fractions containing only further traces of the crystalline lactone.

The not quite pure fractions of the crystalline lactone are sublimed in a small flask under 0.02 mm. pressure and at 2l0230 C. bath temperature. The sublimate gives from acetone-ether directly pure crystals of M.P. 307311 C. (with decomposition.)

Example 1.9 mg. of aldosterone-hydrate are dissolved in 0.05 cc. of purest glacial acetic acid and treated with 0.0165 cc. of 2% chromium trioxide-glacial acetic acid solution (equals 0.33 mg. of CrO A yellow brown precipitate (CrO -complex) is immediately produced which dissolves only gradually. The chromium-trioxide becomes spent after about 2 minutes and then consecutively four equal portions of chromium trioxide solution are further added The first of these portions becomes spent after 5 minutes, the second after 10 minutes and the third after 45 minutes, whereas the fourth is not completely consumed after a further 1 /2 hours. The whole is then evaporated under vacuum at 30 C., treated with Water and 3 drops of 2 N- sulfuric acid and extracted with chloroform-ether (1:3). The separation according to the directions of Examples 3 and 8 yields a trace of acid and 1.8 mg. of neutral constituents, the latter giving from acetone-ether 0.9 mg. of colorless granules of M.P. 298310 C. This compound can likewise be sublimed with-out decomposition under 0.01 mm. pressure and at ZOO-230 C. By a mixed melting point test and a paper chromatog-ram, it is proved to be identical with the lactone described in Examples 3, 8 and 9. The polarity in the B -system of Bush is about the same as or a little higher than adrenosterone. In the formamide-cyclohexane-benzene-(1:1) system at 40 C. R =0.34 (adrenosterone R =0.59).

Example 11 22 to stand for a further 2 hours. Working up according to the directions of Example 8 gives no acid constituents but 7.3 mg. of a neutral crude product. From acetone-ether 6.4 mg. of crystals of M.P. 196199 C. and 0.1 mg. of M.P. 194197 C. are obtained. Recrystallization from acetone-ether gives long, narrow hexagonal leaflets of M.P. 198-200 C. and repeated recrystallization from methanol-ether gives 5.5 mg. of hexagonal (to some extent having rhombic bounding surfaces) leaflets of M.P. 193-194 C.: [a] =+l17.2i3 (c.=0.6186 in chloroform). Reduction test: positive; ultra violet absorp tion: positive. In a paper chromatogram (propylene glycoltoluene) this compound migrates in the same way as 11-dehydro-21 acetyl corticosterone. This compound constitutes the 18,1l-lactone, described in Example 7, of the A 3,20 dioxo-l1B-hydroxy-2l-acetoxy-pregnen-18- acid.

Example 12 20.1 mg. of the 20,18-lactone, described in Example 3, of the 18,11-cyclosemiacetal of A -3,18-dioxo-11fi-hydroxy-etienic acid of M.P. 304-310 C. (with decomposition) are hydrogenated in 2 cc. of glacial acetic acid with 1.75 mg. of previously hydrogenated PtO The absorption of hydrogen is complete after 50 minutes (3.3 cc. H absorbed at 22.4 C. under 731 mm. pressure corresponding to 2.14 mol). The crude hydrogenation product is allowed to stand for 16 hours at 20 C. and in 0.2 cc. of acetic anhydride and 0.3 cc. of absolute pyridine. The whole is then evaporated under vacuum at 40 C., taken up in chloroform-ether (1:3), washed, in each case using 0.3 cc., with dilute hydrochloric acid, water, potassium bicarbonate solution and water (twice), dried over sodium sulfate and evaporated. The residue (20 mg.) gives from a little acetone-ether (about 1:10) 8 mg. of fine needles of M.P. 199-220 C. Purification is best effected by dissolving in acetone-ether (1:1), adding methanol and concentrating to a considerable extent, as a result of which the remaining solvent is practically pure methanol. 4.2 mg. of fiat needles result, which on heating are converted at about 219 C. into thin long needles of M.P. 222226 C. This compound is the 20,1 8-lactone, described in Example 5, of the 18, 11-cyclosemiacetal of 3B-ace'toxy-1LB hyd-roxy-l8-oxo-allo-etianic acid.

Example 13 9.2 mg. of the 20,18-lactone, described in Examples 5 and 12, of the 18,11-cyclosemiacetal of 3,8-acetoxy-11B- hydroxy-18-oxo-etianic acid of M.P. 218-225 C. are heated in a small bomb tube with 0.12 cc. of hydrazine hydrate for 1 /2 hours to 100 C. while the tube is open. Then a solution of 11 mg. of sodium in 0.5 cc. of ethanol is added and the tube is sealed while under mm. pressure and heated for 14 hours to 151 C. After cooling, the alcohol is removed under vacuum and the residue treated with a little Water and extracted by shaking three times, in each case with 10 cc. of chloroform-ether. The extracts washed with water and dried over sodium sulfate leave on evaporation 0.8 mg. of residue (neutral fraction).

The aqueous phase and the wash waters, at 0 C., are brought to pH=1 with hydrochloric acid and again extracted three times by shaking with chloroform-ether (1:3). These three extracts are washed consecutively in counter current, using 0.3 cc. in each case, with the following liquids: water, 2 N-sodium carbonate solution, water, 2 N-sodium carbonate solution, water, water. Drying over sodium sulfate and evaporation give 1.4 mg. of a residue of the 20,18-lactone, described in Example 5, of 3fl',11B,1S-trihydroxy-allo-etianic acid, which compound partially crystallizes.

The sodium carbonate extracts and wash waters are combined, treated at 0 C. with hydrochloric acid until the reaction is acid to Congo red and again extracted with chloroform-ether. The extracts, washed with water and dried over sodium sulfate, give on evaporation 6.7 mg. of residue (acids).

Separation of the acids: The 6.7 mg. of acid mixture gives from acetone-ether in two fractions 4.5 mg. of crystals of melting point 251-268 C. and as a third fraction 0.5 mg. of melting point 235-265 C. As in this case purification is a diflicult matter, the combined crystals mg.) and the residue from the mother liquors (1.7 mg.) are esterified with diazomethane.

The 5 mg. of crystalline acid give 5.1 mg. of crude methyl ester. Recrystallization from acetone-ether yields a mixture of coarse prisms and fine needles. The latter can be washed out with ether-pentane. By careful fractionation and repeated recrystallization 1.7 mg. are obtained of coarse prisms of M.P. 232-237 C. of the 18,11- lactone, described in Example 6, of the 3/3,11;8-dihydroxyallo-etianic acid methyl ester-18-acid. The remaining mother liquors (3.4 mg.) are combined with the methyl ester mixture (1.8 mg.) from the 1.7 mg. of residue from the mother liquors of the crystalline acid. This combined material (5.2 mg.) serves for acetylation (see below).

The 1.7 mg. of the above methyl ester of M.P. 232- 237 C. are allowed to stand for 16 hours at 20 C. with 15 mg. of absolute pyridine and mg. of acetic anhydride. The customary working up yields 1.9 mg. of crude product. From a little acetone with ether crystals are obtained of M.P. 185-194 C. (18,11-lactone of B S-acetoxy-l l 8-hydroxy-allo-etianic acid methyl ester-18-acid).

Separation of the methyl ester mother liquors: The 5.2 mg. of methyl ester mother liquors are acetylated as described above and the acetate mixture (5.6 mg.) chromatographed over 500 mg. of alumina.

The fractions 1-12 (eluted with petroleum ether-benzene rising to 50% benzene content) give only a little residue and at the most traces of crystals.

Fraction 15 (eluted with pure benzene) gives from ether-pentane few flattened, doubly-pointed needles of a secondary product of M.P. 198-201; C. The mother liquor is combined with fractions 13-14 and gives from a little ether with pentane few thick parallelepipeds of M.P. 182-185 C.

Fractions 16-18 (eluted with pure benzene and benzene-ether (95:5) give from ether-pentane 1.018 mg. of pure 18,11-lactone of 3fl-acetoxy-1lfi-hydroxy-allo-etianic acid methyl ester-18-acid of M.P. 190-195 C. and from the mother liquor a further small quantity of the same compound of M.P. 1801:91 C.

Fraction 19 gives a little of a crystal mixture of M.P. 172-186 C.

Fractions 20-26 (eluted with benzene-ether of 10-50% ether content) give from ether-pentane 0.970 mg. of practically pure 3 fl-acetoxy 11;? hydroxy allo etianic acid methyl ester of M.P. 193202 C. and from the mother liquor a further 0.083 mg. of the same compound of M.P. 188-200 C.

Example 14 19.1 mg. of a residue from the mother liquor of the pure 20,18-lactone of the 18,11-cyclosemiacetal of 3;8 acetoxy-lglfl-hydroxy-l8-oxo-etianic acid are heated for 2 hours to 100 C. with 0.3 cc. of hydrazine hydrate and after the addition of mg. of sodium in 1 cc. of ethanol, heated for 17 hours to 151 C. Working up as in Example 13 gives 4.5 mg. of a neutral fraction, 4.4 mg. of the 20,18-lactone, described in Example 5 of 3,8,11fl,18-trihydroxy-allo-etianic acid and 8.5 mg. of acids. The 8.5 mg. of acids are methylated (9.3 mg.) and acetylated (10.2 mg.). The resulting material is chromatographed (40 fractions) over 1 mg. of alumina.

From fractions 13-24 2.7 mg. of crude 18,11 -1actone of 3B-acetoxy-1lfi-hydroxy-allo-etianic acid methyl ester- 18-acid of M.P. 185-193 C. are obtained.

Fractions 25-27 give a little impure 18,11-lactone of 3,8-acetoxy-1lfl-hydroxy-allo-etianic acid methyl ester-18 acid of M.P. 182-192 C.

Fractions 28-38 give 1 .6 mg. of pure 3B-acetoxy-11l3- hydroxy-allo-etianic acid methyl ester of M.P. 192-199 C.

Example 15 5.8 mg. of the 20,18-lactone, described in Examples 5, 13 and 14, of 35,11,6,18-trihydroxy-allo-etianic acid are dehydrogenated with chromium trioxide in glacial acetic acid, until at 20 C. no more chromium trioxide is consumed. The customary working up giv'es 4.4 mg. of a neutral crude product which is chromatographed over 0.3 mg. of alumina. The fractions (0.4 mg.) eluted with ether-methanol (8-50% methanol content) crystallize. Double recrystallization from acetone-ether yields colorless fine needles combined in clusters, of M.P. 275 290 (with conversion into coarse needles) consisting of the 20,18-lactone of 3,1l-dioxo-18-hydroxy-allo-etianic acid of the formula:

What is claimed is: 1. A member selected from the group consisting of a compound of the formula in which R is a member selected from the group consisting of a free hydroxyl group, a hydroxyl group esterified, with a perhalogenated lower alkanoic acid, a hydroxyl group acetalized with an 18-oxo group, a hydroxyl group lactonized with a 13-carboxyl group and an oxo group, R a member selected from the group consisting of a carboxyl group lactonized with an 11-oxy group, an aldehyde group, an aldehyde group acetalized with a lower alkanediol, an aldehyde group semiacetalized with an ll-oxy group and the grouping derived therefrom by lactonization with a 17-carboxyl group, a hydroxymethyl group lactonized with a 17-carboxylic acid group, R a member selected from the group consisting of a free and an esterified carboxylic group and a carboxylic group lactonized with a group in 13-position as said above, X a member selected from the group consisting of a free hydroxyl group, a hydroxyl group esterified with a lower alkanoic acid, an 0x0 group, an oxo group ketalized with a lower alkanediol, and derivatives of such compounds having a double bond extending from the 5- carbon atom.

2. The compound of the formula OC O coon 3 25 3. The compound of the formula A C 0 o H mo 3 4. The compound of the formula 000cm mo J 5. The compound of the formula 6. The compound of the formula 7. (18- 11)-cyclosemiacetal of the compound of the formula 8. The 3-acetate of the compound of claim 6.

9. The compound of the formula 26 10. The methyl ester of the compound of claim 3. 11. The 3,8-acetate of the compound of claim 10. 12. The methyl-ester of the compound of claim 2. 13. The compound of the formula 15. The compound of the formula Simpson et al.: Helv. Chim. Act, volume 37, pages 1200-1223 (June 1954).

LEWIS GOTTS, Primary Examiner.

LESLIE H. GASTON, Examiner. 

14. THE COMPOUND OF THE FORMULA 